The cytochromes P450 (hereinafter abbreviated to P450) constitute a superfamily of membrane enzymes having very varied monooxygenase type activities. Their activities are capable of being used in a wide range of fields of application. There may be mentioned, by way of non-limiting examples:
the bioconversion of a virtually unlimited number of lipophilic molecules by means of reactions of insertion of an oxygen atom, followed or otherwise by rearrangements in carbon--carbon or carbon-hydrogen bonds, and by means of the addition of oxygen to a variety of hetero atoms (sulfur, nitrogen, phosphorus). P450 utilizes aerial oxygen as oxidizing agent. PA1 in vitro diagnosis of the formation of toxic or mutagenic metabolites by human hepatic metabolism of natural or artificial xenobiotic molecules (pollutants, medicinal products, additives). Such prediction is of primary importance, especially in the case of the development of new molecules of pharmaceutical importance. PA1 The identification and destruction of molecules which are toxic or pollute the environment. PA1 too low a reductase/heterologous P450 mole ratio leads to a low specific activity as a result of the lack of reductase. PA1 too high a reductase/heterologous P450 mole ratio leads to the destruction of a considerable fraction of the P450 as a result of the large increase in the number of abortive catalytic cycles and of the production by the excess of reductase of oxygen-containing radicals which are dangerous to the cell. The outcome is a loss of viability of the cells, or even a drop in activity due to the destruction of cytochromes P450. PA1 too low a cytochrome b5/P450 mole ratio leads to a reduced activity of class B P450. Moreover, the presence of a high level of cytochrome b5 appears to exert a protective effect against the toxic effects linked to excessive reductase levels; PA1 too high a cytochrome b5/P450 mole ratio leads to the inhibition of class A P450 activity, and can potentially reduce the amount of unbound intracellular heme and thus be toxic to the cell. PA1 by the construction of fusion proteins artificially combining an NADPH-cytochrome P450 reductase (homologous or heterologous) and the cytochrome P450 itself on the same polypeptide chain. PA1 by the use of a vector carrying both an expression unit for the P450 reductase and an expression unit for a heterologous P450 on the same plasmid. PA1 the first is that it does not permit an adjustment of the ratio of the amount of reductase to that of the P450. Since the reductase has a priori a much faster catalytic cycle than the cytochrome, the optimum ratio for the activity is not necessarily 1:1, and can vary from one P450 to another, or even from one substrate to another for the same P450. PA1 the second stems from the poor yield of synthesis (or from poor stability) of the fusion protein; this manifests itself in most cases in a large reduction in the level of expression of the fusion protein relative to that of the P450 expressed alone. This decrease causes most, if not all, of the gain in specific activity resulting from the fusion to be lost in terms of yield. PA1 the third defect is the impossibility, if not technical, then at least practical, of generalizing this approach to the fusion of more than two proteins. PA1 (a) inactivation by mutation of one of the heterologous genes. Such an event is generally selected spontaneously as a result of the toxicity to the cell of the heterologous genes or at least of the associated activities. PA1 (b) deletion by recombination of the heterologous gene on duplication of the wild-type gene (conversion) within a heterozygous locus (see Diagram 1). The selective advantage is twofold, since it has both an identical effect to (a) and an effect which leads to a wild-type homozygous context. PA1 (c) elimination of one of the heterologous genes, or evolution of important characteristics of the strain (linked to the rearrangement of unknown endogenous heterozygous genes which are not directly linked to the heterologous genes but have significant functions for the activities of interest) by a meiotic recombination which might occur at the heterozygous loci and which would be followed by sporulations and then by spontaneous crosses. PA1 the strains of this set are completely isogenic with one another, with the exception of the specifically modified loci, in particular the mating-type locus; PA1 an NADPH-cytochrome P450 reductase and a cytochrome b5 are produced in each cell, at a homogeneous level independent of the level and nature of the heterologous cytochrome P450 possibly expressed; PA1 the total level of reductase and of b5 is adjustable by modification of the composition of the culture medium, independently of the level of heterologous P450 possibly expressed; PA1 the maximum total level of expression of the reductase and of b5 can be at least equal (in molar terms and at maximum induction) to that of the heterologous P450 produced; PA1 the minimum levels of the reductase and of heterologous b5 which can be produced (under non-induction conditions) are at least 10-fold lower than the maximum level of the reductase and of b5 defined above; PA1 the reductase may be overproduced independently of the cytochrome b5; PA1 these strains carry the selectable markers needed for the use of common plasmids, and are, independently of the above properties, "good hosts" for the expression of heterologous cytochromes P450. PA1 the elements PES1-1 and PES1-2 are not in themselves novel, since they are, respectively, the haploid mating type a and alpha forms of S. cerevisiae yeast strain W303.1B.
In view of this broad field of application, the expression of heterologous forms of P450 and of their activities in microorganisms which lack them clearly merits attention. Two problems then became apparent: first, the membrane nature of the P450 of eukaryotes makes it desirable to use a host microorganism of the eukaryotic type, leading to the choice of yeasts; second, these enzymes are functional only in the presence of "associated enzymes" which play the part of specific electron transporters. Whereas the diversity of cytochromes P450 is extreme, the "associated REDOX enzymes" are few in number and comprise cytochrome b5, NADH-cytochrome b5 reductase and, most especially, NADPH-cytochrome P450 reductase. Moreover, certain enzymes, termed phase II, such as, for example, microsomal epoxide hydrolase, may be needed for the metabolic coupling between different P450's, or for the destruction of highly reactive chemical intermediates formed during certain reactions, such as, for example, the metabolism of polycyclic hydrocarbons.
The high level in vivo expression of heterologous cytochromes P450 in yeast leads to high levels of activity only if these "associated redox enzymes", which are essential to the functioning of P450, can be coexpressed in the yeast, in suitable stoichiometries relative to one another and to the P450. Recent data show that,
In order to solve these problems, there was constructed according to the invention, by genomic integration of synthetic genes, a set of yeast strains expressing, stably and in a manner which can be modulated (by the composition of the culture medium), an enzymatic environment which can be optimized for expression of the activity of any heterologous P450.
Many publications have described the expression of heterologous cytochromes P450 in yeast. Nevertheless, the problem of optimization of the cellular environment to permit high activity in vivo has seldom been tackled, the proteins produced often being used for analytical purposes or purposes of in vitro research.
The problem of optimization of the activities has been tackled hitherto in two ways:
Both of these systems, namely the construction of fusion proteins (P450--P450 reductase) or of plasmid coexpression vectors, possess serious drawbacks:
1--the construction of fusion proteins (P450-reductase) is a lengthy operation which is difficult to optimize (except empirically by a trial-and-error approach), in the present state of knowledge, in respect of the molecular design of the fusion. This is not a priori a genuine defect when attention is focused on a single activity and when lengthy research can be devoted to it; it is, however, a serious weakness during a process of development where several types of activity have to be tested in a reasonable time in order to adjust the tool to the problem. Apart from this fact, this system possesses three major defects which are inherent in it: PA0 2--The coexpression of several heterologous proteins from a single plasmid appears at first sight to be a good approach. Nevertheless, such large plasmids are potentially unstable genetically, most especially (and this is most often the case) when the same promoter elements are used for the different genes. This instability is a definite problem for an industrial application. PA0 . . XXX . . . YYYY . . . ZZZ.fwdarw. . . . XXX . . . YYYY . . . ZZZ . . . PA0 . . XXX . . . hhhhh . . . ZZZ .fwdarw. . . . XXX . . . YYYY . . . ZZZ . . . PA0 XXX and ZZZ : wild-type intergenetic sequences PA0 hhhhhh : heterologous gene PA0 YYYYY : wild-type gene which is the target of the integration PA0 1--the yeast strain is a diploid strain in which the alleles are isogenic, with the exception of the loci which carry the heterologous genes and of the mating-type locus, which are heterozygous, PA0 2--the heterozygous loci lack an allele carrying a wild-type gene, and PA0 3--the heterologous genes are placed under the control of an inducible and regulable promoter. PA0 1--the genomic sequences of PES1-2 yeast lying between the first Kpn1 site located upstream of the endogenous coding frame of NADPH-cytochrome P450 reductase and the first codon of this same coding frame are removed. PA0 2--the sequences removed are replaced, starting from the Kpn1 (half-site) and in the following order, by: the gene encoding orotidine-5-phosphate decarboxylase (of S. cerevisiae), a fragment of the GAL10 gene of S. cerevisiae), a fragment of the CYC1 gene of S. cerevisiae comprising the transcription initiation sequences of this gene and a few nucleotides of synthetic sequence. PA0 1--the genomic sequences lying between the Kpn1 half-site immediately preceding the functional URA3 gene of PES1-3 and the first BspMI site encountered on the 5' side (relative to the orientation of the coding frame of the reductase gene) of this position (formerly in the promoter portion of the wild-type NADPH-cytochrome P450 reductase gene) are removed. PA0 2--the whole of the NADPH-450 reductase coding frame (starting from the BamHI site) together with its flanking portion on the 3' side as far as the first BspMI site encountered on the 3' side (same convention as above) is removed. PA0 3--The portion removed is partially replaced, in the following order and starting from the end where the BspMI site encountered on the 3' side was located and in the following order: the gene encoding orotidine-5-phosphate decarboxylase (of S. cerevisiae), a fragment of the GAL10 gene of S. cerevisiae, a fragment of the CYC1 gene of S. cerevisiae comprising the transcription initiation sequences of this gene followed by a few nucleotides of synthetic sequence, and by the complete coding frame of the human cytochrome b5 gene, followed by a short synthetic sequence, followed by a fragment of the phosphoglycerate kinass gene of yeast and comprising the 3'-flanking sequences of this gene. PA0 4--The mating-type sign of the strain is changed from alpha to a. PA0 The strain PSE1-34 consists of the diploid of the haploid strains PSE1-3 and PSE1-4. PA0 The strain PSE1-31 consists of the diploid of the haploid strains PSE1-3 and PSE1-1. PA0 The strain PSE1-42 consists of the diploid of the haploid strains PSE1-4 and PSE1-2. PA0 The strain PES1-12 consists of the diploid of the haploid strains PSE1-1 and PSE1-2.
Moreover, the multicopy plasmids used, as a result of the random distribution of the number of copies in each cell, lead to cell populations which are very heterogeneous as regards the level of expression of each of these two enzymes (reductase and P450). The mono-oxygenase activity obtained (which is in practice the useful value) is then the mean of a square-law distribution; this value is bound, for obvious reasons, to be lower than the activity value which would be achieved if the P450 (even on a multicopy plasmid) were expressed in a cellular context having a homogeneous and high level of reductase.